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Journal of Applied Biotechnology Reports
Journal Prestige (SJR): 0.134
Number of Followers: 1  

  This is an Open Access Journal Open Access journal
ISSN (Online) 2322-1186
Published by Baqiyatallah University of Medical Sciences Homepage  [5 journals]
  • Direct Detection of Phanerochate chrysosporium, Engyodontium album, and
           Fusarium venenatum in Soil Samples Collected from Different Regions of
           Iran by PCR Method

    • Abstract: Introduction: Engyodontium album, Fusarium venenatum, and Phanerochaete chrysosporium fungi have an important role in the production of proteinase K, Quorn mycoprotein, and bioremediation, respectively. There are several techniques for the detection of fungi in soil. The purpose of this study was to detect and identify E. album, F. venenatum, and P. chrysosporium, directly from soil using the Polymerase Chain Reaction (PCR) method.Materials and Methods: A total of 240 soil samples were collected from different regions of Iran, including Tehran, Zanjan, Hamadan, Kermanshah, Kurdistan, and Hormozgan Provinces. The DNA was extracted and purified directly from soil samples with the modified phenol-chloroform method and by PVPP (Polyvinylpolypyrrolidone) column, respectively. The PCR method was performed using designed specific primers. The PCR products of the E. album, F. venenatum and P. chrysosporium with an approximate size of 248, 202, and 502 bp were sequenced, respectively.Results: In this study two isolates of P. chrysosporium, 1 isolate of F. venenatum, and 1 isolate of E. album were identified. The fungi detected with specific primers in soil samples were compatible with the results of sequencing.Conclusions: This investigation described a reliable method that can be used to detect important fungi in the industries and biotechnology directly in soil using specific primers. The results can provide an appropriate platform for next applied research and mass production of valuable fungal products in industries and biotechnology. 
  • A Gene Expression Signature to Predict Chemotherapy Response of Colorectal
           Cancer Patients: Systems Biology Analysis on Transcriptomics Data and
           Experimental Validation

    • Abstract: Introduction: Gene expression profiling has high potential in the identification of diagnostic, predictive, and therapeutic gene targets in human cancers such as colorectal cancer (CRC).  Accordingly, in this study, an integrated systems biology analysis was done on several microarray datasets to identify key genes involved in CRC chemoresistance and also to differentiate peoples who benefit from chemotherapy. Subsequently, the findings were validated experimentally. Materials and Methods: Datasets were retrieved from Gene Expression Omnibus (GEO). Gene expression analysis was performed using the ExAtlas software. Gene enrichment analysis was done using g: profiler. Protein-Protein Interaction Network (PPIN) was constructed in STRING and visualized/analyzed by Cytoscape 3.8.0. Significant modules were identified using the MCODE plugin in Cytoscape. The clinical importance of candidate genes was evaluated using ROC analysis and immunohistochemistry. Key candidate genes were validated using Real-Time  PCR. Results: According to findings, 26 datasets were selected. Gene expression analysis revealed 6463 Differentially Expressed Genes (DEGs), among which 4323 were unique and 2140 were related to overlapping DEGs between datasets. The overlapping DEGs with at least four shared datasets (n=217 DEGs) were selected for further analysis. Overlapping DEGs were mainly enriched in the cellular process of response to chemicals stimulus. Most selected DEGs were enriched in KEGG pathways of cancer Benzo(a)pyrene metabolism and glucocorticoid receptor signaling. Fourteen hub genes and two significant modules were identified. Six hub genes (candidate genes) were contributed in significant modules. Among candidate genes, LCN2, CXCL8, and EGR1 expression were significantly associated with chemotherapy response of CRC patients and chemosensitivity of CRC cell lines (P<0.05). Conclusion: This study revealed three genes signature for predicting chemotherapy responsiveness and treatment decision-making in CRC patients and also for therapeutic purposes. 
  • Medicinal Applications, Chemical Compositions, and Biological Effects of
           Algerian Ocimum basilicum L.var Genovese with the Conversion of
           Experimental Doses to Humans

    • Abstract: Introduction: This paper aims to analyze the medicinal uses of Ocimum basilicum L.var Genovese (basil) in western Algeria and its effectiveness.Materials and Methods: For the experiments, 154 structured questionnaires were collected to list the medicinal uses of basil. The essential oil of O. basilicum (EOB) obtained by hydro-distillation was analyzed by the GC/MS. The ethanolic and aqueous extracts (EEB and AEB) were analyzed by HPLC. The antioxidant activity was measured by DPPH assays and the antimicrobial activity was measured against five microbes. For the in vivo study, Swiss albinos mice were used to determine the toxicity using Lorke’s method. The anti-inflammatory activity was determined using the Carrageenan method. The experimental doses were converted from mice to humans using the Km factor.Results:  The ethnobotanical study indicates that local people use basil to treat diseases and health problems (50% for inflammation and 38.11% for microbial diseases). The results also show that EOB contains 41.3% linalool, whereas ethanolic extract contains benzoic acid (50.86 mg/g). The IC50 value is 556, 878.7, and 962.3 µg/ml for EOB, EEB, and AEB, respectively. The EOB and AEB inhibit the positive Gram bacteria and yeast; the EEA inhibits the negative Gram. The LD50 is 400, 470, and >5000 mg/kg for AEB, EOB, and EEB respectively. The results of the anti-inflammatory test highlight 76.33, 71.0, and 60.43% inhibition of edema at a 100 mg/kg dose for EOB, AEB, and EEB, respectively.Conclusion: The Algerian basil can be considered as an antioxidant, antimicrobial and anti-inflammatory.
  • Influence of Extracellular Vesicles from Lactobacillus rhamnosus GG on the
           Cell Adhesion and mmp2 and mmp9 Genes Expression in Colorectal Cancer

    • Abstract: Introduction: Lactobacillus rhamnosus GG is a probiotic bacterium with anti-cancer and anti-microbial characteristics. In addition, Extracellular Vesicles (EVs) from Lactobacillus rhamnosus GG showed apoptotic impacts on colorectal cancer cells. In this study, we aimed to isolate EVs from Lactobacillus rhamnosus GG (EVL) and identify its effect on cell adhesion and expression of mmp2 and mmp9 genes.Materials and Methods: HT29 cells were exposed to 16, 32, 64, 128, 256, 512, 1024, and 2048 µg/ml EVL, and the cell viability, cell adhesion, and mRNA gene expression were investigated using the MTT assay, cell adhesion assay, and real-time PCR.Results: We found that 128, 256, 512, 1024, and 2048 µg/ml of EVL led to attenuated cell viability of HT29 cells (p<0.05). Moreover, the expression of both mmp2 and mmp9 genes and cell adhesion significantly reduced at 1024 and 2048 µg/ml EVL compared to untreated cells (p<0.05).Conclusions: According to the findings of the present study, it is suggested that EVL could be effective in cancer cell cytotoxicity. 
  • Prediction of the Most Deleterious Missense Variants of Human Somatostatin
           Gene by Combining Computational Algorithms, Molecular Docking and Dynamic

    • Abstract: Introduction: Somatostatin (SST) is a versatile hormone that plays a key role in inhibiting the secretion of several pituitary hormones. It is well known that SST participates in the regulation of other essential proteins throughout the body. The abnormal function of the SST protein is still not fully understood. In this study, the disease susceptible Single Nucleotide Polymorphisms (SNPs) in SST were classified by using different computational algorithms.Materials and Methods: Sequence-based and structure-based computational tools were employed to classify the most disease susceptible nsSNPs that would have the most harmful effects on SST protein. Docking and molecular dynamic simulations were performed to compare the ability of the normal SST and its most deleterious mutants to bind with corresponding SSTRs to assess the potential role of these nsSNPs to alter protein-protein interactions.Results: Two nsSNPs, namely L13P, and G104S, were considered to have the most severe functional consequences on the 3D structure of SST. These results were confirmed by molecular dynamic simulations. Docking of SST and its mutant models with SST receptors (SSTR1-SSTR5) showed remarkable roles of both mutant L13P and G104S in altering the binding of SST with SSTR2 and SSTR5.Conclusions: The findings of the present study provide the first comprehensive in silico prediction for assessing the damaging effects of nsSNPs on SST, which may help in a better understanding of how the altered SST would impact the overall health of the body. This study may provide a platform to conduct large-scale experiments on the genetic polymorphism of SST. 
  • Comparable Analysis of Mature Antigen-Loaded Dendritic Cells Preparation
           Methods for Cytokine-induced Killer Cells Activation in vitro

    • Abstract: Introduction: Colorectal cancer remains a leading cause of cancer-related mortality worldwide. Cancer immunotherapy involves autologous tumor-derived antigen-loaded Dendritic Cells (DCs) that activate potent antitumor immunity. Cytokine-Induced Killer (CIK) cells are a heterogeneous population of anti-tumor cytotoxic CD8+ T-cells. Cancer immunotherapy using a combination of CIK cells with DCs vaccines is a promising strategy. We investigated some of the latest developments in the DCs vaccination field, with a special emphasis on strategies to prepare highly immunogenic tumor cell antigens to load and to activate DCs. In this context, we applied the effects of immunogenic treatmentmodalities (heat shock) and four potent inducers of immunogenic cell death and apoptosis (mitoxantrone, oxaliplatin, 5 fluorouracil, and staurosporine) on DCs biology and their employment in DC-based activation of CIK cells.Materials and Methods: DCs were generated from bone marrow cells using granulocyte-macrophage colony-stimulating factor and Interleukin (IL)-4. CIK cells were expanded by interferon-gamma (IFN-γ), anti-CD3 monoclonal antibody, and IL-2 stimulation. The cytotoxic activity of CIK cells against CMT-93 cancer cells was assessed by MTT assay. IFN-γ production of CIK cells was examined by ELISA.Results: Coincubation of untreated DCs with CIK cells significantly induces antitumor immunity of CIK cells. Mature DCs loaded by polyethylene glycol with mitoxantrone-oxaliplatin-induced apoptotic tumor cells stimulate greater cytotoxicity of CIK cells compared to DCs loaded with staurosporine, oxaliplatin- and 5-fluorouracil-inactivated tumor cell antigens, whole tumor lysates, and total tumor RNA in CMT-93 cells.Conclusions: Heat shock and mitoxantrone-oxaliplatin-treatment are the best approaches for cancer cell antigens preparation for DC-induced CIK cells activation. 
  • Isolation and Purification of Antifungal Compounds from the Green
           Microalga Chlorella vulgaris

    • Abstract: Introduction: The current study aimed to purify antifungal compounds from Chlorella vulgaris extracts, fractions, sub-fractions and pure compounds against different strains of mycotoxigenic fungi.Materials and Methods: Antifungal activity was conducted using disc diffusion assay, TLC-bioautography and Minimum Inhibitory Concentration (MIC). Isolation, purification and structure elucidation of antifungal compounds were carried out using column chromatography, Thin Layer Chromatography (TLC), UV-Vis spectrophotometer, Gas Chromatography–Mass Spectrometry (GC-MS), and Nuclear Magnetic Resonance (NMR).Results: C. vulgaris Diethyl Ether Extract (DEE) showed the highest antifungal activity against all tested fungi with inhibition zone from 11.5 to 21.9 mm. By fractionation of DEE, Fraction F3 (chloroform:methanol, 50:50) and F5 (methanol 100%); sub-fraction CF3-10 and CF5-10 exhibited antifungal activity against all tested fungi. Two pure compounds, hydroxyphenophytin B and hexadecanoic acid methyl ester, with antifungal activity were isolated from CF3-10 and CF5-10, respectively.Conclusions: C. vulgaris DEE and isolated compounds can be used as promising antifungal agents from natural sources against mycotoxigenic fungi at post-harvest or storage stages. 
  • Pigment Productions by Spirulina platensis as a Renewable Resource

    • Abstract: Introduction: Recently, Spirulina platensis has scientifically become popular because of its importance as food, feed, and a natural producer of pigments with specific nutritional and functional characteristics.Materials and Methods: In this study, the effect of various environmental factors affecting growth conditions of Spirulina platensis, including primary inoculation, light-dark cycle, cultivation time, Light-Emitting Diode (LED) composition, nitrogen source, carbon source, and NaCl concentration, on biomass, C-phycocyanin (C-PC), Allophycocyanin (APC) and chlorophyll-a contents were assessed using Placket-Burman Design (PBD).Results: Results showed that out of the seven screened factors, four factors of carbon source, LED composition, light-dark cycle and NaCl concentration significantly affected biomass production (p<0.01). Among the investigated factors, nitrogen source, light-dark cycle, and NaCl concentration had significant effects on phycocyanin production (p<0.05). Results showed that cultivation time, light-dark cycle, and NaCl concentration significantly affected the production of allophycocyanin (p<0.05). Furthermore, NaCl concentration, carbon source, LED composition, cultivation time, and initial inoculation included significant effects on chlorophyll-a production (p<0.05).Conclusions: The present study screened variables affecting biomass, phycocyanin, allophycocyanin, and chlorophyll-a production as the first step in optimizing Spirulina platensis growth condition. Briefly, NaCl concentration was one of the factors which had a significant impact on all responses. The dark cycle also had an effect on three dependent variables except for chlorophyll-a production. 
  • Detection of Some Enteric Bacterial Toxin Via Modified ELISA Assay

    • Abstract: Introduction: Heat-labile enterotoxin (LT) of Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin (Stx) of Shigella dysenteriae serotype 1 are two important toxins of food-borne pathogens associated with diarrheal disease. These agents have been recognized as the first leading causes of neonatal diarrhea and the Hemolytic-Uremic Syndrome (HUS). These toxins have two subunits, A and B, that B subunit can be used as a diagnostic tool.Materials and Methods: In this study, the LTB and STXB genes were amplified by using the Polymerase Chain Reaction (PCR) technique and cloned into the prokaryotic expression vector. Following the expression of recombinant LTB and STXB proteins, mouse polyclonal anti recombinant LT enterotoxin and Shiga toxin B subunits were produced for immunological detection. An Enzyme-Linked Immunosorbent Assay (ELISA) was developed for detecting toxins using clinical samples.Results: Our results showed that the competitive ELISA has high specificity. In addition, the detection limits for LTB and STXB were 20 ng and 90 ng, respectively.Conclusions: Our findings revealed that the B subunit of LT and STX can be of a great help in detecting these agents. 
  • Root Protein Interactomics of Salt Stress-Induced Proteins of Wheat
           Genotypes KH-65 (Salt-Tolerant) and PBW-373 (Salt-Susceptible)

    • Abstract: Introduction: Wheat crop is moderately tolerant to salt stress and is considered as an excellent system to study salt stress tolerance despite its genetic complexity. In the present study, the top ten biological processes in a root proteome were mapped for Protein-Protein Interaction (PPI) networks and analyzed to examine the effect of salt stress on wheat cultivars KH-65 and PBW-373.Materials and Methods: NaCl salinity treatment 0 and 300 mM NaCl was performed on a three-leaf stage plant. Roots proteins were analyzed by liquid chromatography-mass spectroscopy. Proteins were grouped according to GENE ontology terms for biological processes and arranged in descending order. Interactome analysis was done through the STRING database.Results: Interacting root proteins of tolerant line KH-65 show a comparatively higher number of nodes, edges, and interacting proteins than the sensitive line, PBW-373. The number of proteins whose expression was positively induced upon salinity stress was significantly higher in the roots of salinity-tolerant KH-65 than that of the PBW-373 roots. Moreover, the fold induction too was also high in the tolerant line. Similarly, the number of participant proteins in an interaction network of the KH-65 roots was higher than that of the PBW-373 cultivar.Conclusions: This analysis provides valuable information in elucidating the molecular mechanism associated with salt stress response in wheat seedlings’ roots. The observation is correlated with the efficient salt tolerance capacity of KH-65. The higher expressing proteins in interaction networks may be seen under the increased salt tolerance capabilities of salt-tolerant KH-65 line. 
  • The Effects of Royal Jelly on the Pro-Inflammatory Innate Immunity
           Cytokines in Patients Infected with Hepatitis B Virus and Its Antiviral

    • Abstract: Introduction: Innate immunity cytokines conduct significant functions in the stimulation and induction of liver complications in Hepatitis B infected patients. It has been reported that Royal Jelly (RJ) has important roles in decreasing the pro-inflammatory cytokines in both in vitro and in vivo conditions. This project aimed to investigate the impacts of 1 month RJ administration on interleukin 1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6) serum levels in the Hepatitis B patients.Materials and Methods: In this research, 30 Hepatitis B cases (patients) were entered in order to be under the treatment of RJ for 1 month. Before and after treatment with RJ, Hepatitis B Virus (HBV) copy numbers were evaluated using Real-Time PCR and IL-1β, TNF-α, and IL-6 serum levels were evaluated using the ELISA technique.Results: The RJ treatment significantly reduced the number of HBV-DNA copies and led to down-regulation of TNF-α and IL-6, which were not substantial. The IL-1β, TNF-α, and IL-6 serum levels were not changed after RJ treatment in both men and women.Conclusions: Based on the findings of the present study, it seems that RJ plays anti-viral and anti-inflammatory roles in the in vivo conditions in infected patients with HBV. 
  • The Association of -1031 T/C TNF-α Gene Promoter Polymorphism with the
           Incident of Gastric Carcinoma Among Iraqi Patients

    • Abstract: Introduction: Among all the different types of cancer, Gastric Carcinoma (GC) has become the most frequently diagnosed and has continued to be a major public health issue in the last few decades worldwide. Association between the polymorphisms of the (T/C) -1031 TNF-α promoter gene sequence (rs1799964) and the incidence of gastric carcinoma was tested in Iraqi patients undergoing 5-FU plus Cisplatin, patients without chemotherapy, and healthy controls.Materials and Methods: Blood samples were collected from patients and control groups to carry out the molecular and immunological diagnostic tests. Two ml of blood were collected in EDTA tubes for (T/C) -1031 TNF-α genotyping by using the RFLP technique. The serum part was used for the purpose of immunological tests via ELISA technique.Results: Findings revealed that the homozygous wild genotype (T/T) was more abundant than other genotypes (T/C and C/C) in different groups of this study (82, 73, and 72% in control, patients under treatment, and patients without treatment respectively). From the Chi-square (data of the p value), there were no significant differences between genotypes in the different groups. TNF-α concentration increased significantly in heterozygous (T/C) and homozygous (T/T) genotypes in patients without treatment (p = 0.0143) and was highly significant in healthy control samples (p = 0.0003). The results showed there were non-substantial differences (p = 0.1083) in the TNF-α concentrations between different genotypes in patients treated with chemotherapy.Conclusions: The genotyping study through the RFLP Technique and allele frequency measurement revealed that the homozygous wild genotype (T/T) was more frequent compared with the other genotypes in different groups of this study. However, TNF-α concentration significantly increased in heterozygous (T/C) genotypes. Non-significant differences in TNF-α concentration were detected among different genotypes in gastric carcinoma patients under treatment of chemotherapy. 
  • Phosphorylated H2AX: Prospective Role in DNA Damage Responses and a
           Credible Tool for Translational Cancer Research

    • Abstract: Double Strand Breaks (DSBs) are the most deleterious DNA lesions among the first signs of cancer in eukaryotic cells. Occurring on exposure to one or more endogenous and/or exogenous factors and leading to fatal consequences like chromosome aberrations and genomic instability. Therefore, programmed and coordinated cellular processes function intra-cellular to stabilize the genomic information and repair damages. These processes are primarily a part of DNA replication and cell-cycle progression. However, various signaling factors also activate specialized DNA Damage Response (DDR) pathways for repairing DSBs. Lately, the phosphorylated histone variant H2AX (γH2AX) has been identified as a biomarker for DNA damage. Studies have shown a correlation between the concentration of cellular γH2AX and the extent of DNA damage. Hence, the quantification of DNA lesions can be done using simple spectroscopic and radiological techniques or immunofluorescent staining. For this reason, γH2AX has especially gained value as a biomarker in translational cancer research. Moreover, this approach may act as a boon in clinical trial studies for understanding the different phases of cancer and studying the pharmacodynamics of prospective drugs. Recently, γH2AX based studies have indicated the indispensable fate of DNA damages occurring during normal neurological development and in disorders like obesity. The current review focuses on the role of γH2AX in DDR pathways, and ways in which the correlation of γH2AX and DNA damage can be applied in monitoring the clinical response of DNA targeted therapies.
  • Study of Cannabinoids Biosynthesis-Related Genes in Hemp (Cannabis sativa
           L.) under Drought Stress by In Vitro and In Silico Tools

    • Abstract: Introduction: Cannabinoids can be found as the specific secondary metabolites of hemp (Cannabis sativa L.), including Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabichromene (CBC). There are many enzymes, particularly cannabichromene synthase, cannabidiolate synthase, and Δ9-tetrahydrocannabinolate synthase, contributing to the biosynthesis of the cannabinoids. Environmental stress, particularly drought, can induce secondary metabolites. In the present study, we have tried to investigate and understand the key factors such as drouth-induced Transcriptional Factors (TFs) involving in the pathway by employing in vitro and in silico tools.Materials and Methods: After providing the genes' names and IDs from the National Center for Biotechnology Information (NCBI), Transcription Start Sites (TSS) and TATA-box were predicted by the TSS Plant website, as well as involved transcriptional factors. The expression of the genes was assayed under drought conditions by in silico and in vitro tools, R software, and Real-time PCR, respectively.Results: The findings identified all the genes contributing to biosynthesis cannabinoids in drought conditions. There were actually six TF sites and four TF sites for the gene of olivetolic acid cyclase and AAE1, respectively.Conclusions: Drought stress can induce overexpression of the genes encoding B3 domain-containing proteins, MLP28, MYB binding site, transcriptional repressor OFP7, and WAK1 as TFs respond to biotic and abiotic stresses in Cannabis sativa plants.
  • Surface Modification of Superparamagnetic Iron Oxide Nanoparticles by
           Argon Plasma for Medical Applications

    • Abstract: Introduction: Hyperthermia is rapidly becoming a clinical reality as a tool for the treatment of malignant disease but there are some major problems in the way of using superparamagnetic nanoparticles coated with polymer in medical applications. Modifying the magnetic nanoparticles without using surface coating is more appropriate. In this study, we presented a new physical technique, by surface treatment of nanoparticles with argon gas plasma, to modify the surface of nanoparticles for improving their crystal structure and magnetization.Materials and Methods: In this study, Fe3O4 nanoparticles were synthesized using the co-precipitation method. The nanoparticles were then treated with plasma in a vacuum chamber. In this method, the Radio Frequency (RF) generator 13.56 MHz was used as a power supply and the plasma treatment was applied for 10 and 15 min.Results: Due to the decrease in surface irregularities, the nanoparticle aggregation decreased and their colloidal stability increased. Moreover, with Value Stream Mapping (VSM) analysis the magnetism of the nanoparticles improved along with an increase of plasma power and plasma treatment time due to the reduced crystal defects and crystal growth. By using the AC magnetic field generator with a frequency of 92 kHz and amplitude of 125 Oe, results show that along with an increase of plasma power and plasma treatment time due to the increased magnetization and colloidal stability, heat generation by these nanoparticles increased in a ferrofluids system in the presence of AC magnetic field. In addition, the locking temperature of nanoparticles has also increased.Conclusions: Our results suggest that the surface modification of Fe3O4 nanoparticles, using plasma treatment, is an appropriate candidate for some medical applications such as magnetic resonance imaging, drug delivery, and especially for magnetic hyperthermia.
  • Evaluation of Pomegranate Seed Extract and TGF-β3 on Chondrogenic
           Differentiation of Human Adipose-Derived Stem Cells

    • Abstract: Introduction: The repair of the cartilage continues to be a big challenge. Autologous cartilage is the gold standard, but enough sources are not available. The development of stem cells, biomaterials, and bioactive factors has led to cartilage tissue engineering becoming a promising strategy for the regeneration of cartilage defects. In this study, the effect of Transforming Growth Factor Beta 3 (TGF-β3) and Pomegranate Seed Extract (PSE), as chondrogenesis inducers, were evaluated.Materials and Methods: Human Adipose-Derived Stem Cells (ADSCs) were seeded in alginate scaffolds and cultivated for two weeks in chondrogenic media. Finally, the MTT assay was used to test the effect of TGF-β3 and PSE on the survival of differentiated cells. The mRNA levels of the cartilage-specific markers such as SRY-box9 (SOX9), Collagen type II (COLII), Collagen type X (COLX), and Aggrecan (ACAN) were determined by RT-PCR. Also, CD markers were evaluated by flow cytometry.Results: Using both natural PSE and chemical TGFβ3 inducers simultaneously, had the best results in chondrogenesis by increasing the expression of the SOX9, COLII, and ACAN genes. Furthermore, the flow cytometry analysis indicated that the expression of CD14 marker of differentiated cells significantly increased, although the expression of CD44 marker decreased two weeks post differentiation.Conclusions: PSE is a suitable inducer and its combined use with TGF-β3 can improve the efficiency of the chondrogenic potential of ADSCs. Our results suggest that PSE may have the potential to be used in tissue engineering as a replacement for TGF-β3. 
  • Evaluation of Chlorella sorokiniana Biomass Recovery by Using Different
           Chemical-based Flocculants

    • Abstract: Introduction: The nature and the concentration of the chemical agents responsible for cell flocculation are the bottlenecks for microalgae recovery. The aim of the present study was to evaluate different chemical-based flocculants for Chlorella sorokiniana flocculation.Materials and Methods: The biomass recovery efficiency was evaluated by comparing self-flocculation and flocculation with the ferric chloride, sodium hydroxide, aluminum sulfate, and zinc sulfate. After identifying the best flocculating agent, its concentration was varied to determine the optimal condition by rapid agitation followed by sedimentation (0.25 to 1 g/L).Results: Zinc sulfate was unsuitable for this strain due to an efficiency lower than 40%. Self-flocculation and sodium hydroxide were fairly efficient (48.65% and 58.06%, respectively). Aluminum sulfate produced moderate results (56.27%), but flocculation took a long time to become efficient. Ferric chloride showed the best potential for flocculation, and in the analysis of different concentrations (0.25 to 1 g/L) showed to be fast and efficient (nearly 80% of biomass recovery in 10 min) at a concentration of 0.75 g/L.Conclusions: All the flocculants tested in this study can be utilized for biomass recovery, except for the zinc sulfate. The procedure was efficient, inexpensive, and contaminant-free for the recovery of C. sorokiniana biomass. 
  • Optimization of Degradation of Petroleum Crude Oil by Lysinibacillus sp.
           SS1 in Seawater by Response Surface Methodology

    • Abstract: Introduction: The prevalence of petroleum oil spills in oceans and seas is on the rise in India, resulting in widespread detrimental effects on the environment. Bioremediation by bacteria is an eco-friendly and safe technique for the removal of these pollutants from seawater.Materials and Methods: An indigenous bacteria, isolated from garage soil was grown on Bushnell Agar plate with Petroleum Crude Oil (PCO) as a carbon source. It was identified by biochemical characterization and 16S rRNA sequencing. The effect of factors such as concentrations of PCO, inoculum and glucose, agitation speed, pH, and degradation time on the growth of bacteria and PCO degradation in seawater was studied by one factor at a time approach. Screening and optimization were performed by Factorial Design and Central Composite Design respectively.Results: According to findings, isolated bacteria degraded PCO within 48 h and could decolorize 6-dichlorophenol indophenol within 36 h. It was identified as a novel Lysinibacillus sp. SS1, which grew in the pH range of 4.0 to 10.0 and tolerated salinity of 6.0% w/v. Significant factors (concentrations of glucose, inoculum, and pH) were optimized and optimum levels were 11.7% v/v inoculum, 11.36 g/L glucose, and pH 8.6. Maximum degradation of 84 ± 0.13% was achieved when grown in seawater supplemented with 4.0% v/v PCO, at 27 ± 2 °C at 80 RPM in 28 days at optimized conditions.Conclusions: The present study is the first study reporting optimization of degradation of PCO by Lysinibacillus species. Lysinibacillus sp. SS1 could effectively degrade petroleum hydrocarbons in extreme conditions of seawater and can be applied for the treatment of oil spills. 
  • All-in-One Molecular Cloning as a New Gene Manipulation Method

    • Abstract: Introduction: DNA cloning plays a crucial role in biotechnology laboratories and biotechnology-related fields, which facilitates the primary yet crucial step for further studies in molecular biology. Many laboratories worldwide still use restriction enzyme-based cloning methods to construct expression vectors owing to financial constraints, time-consuming, and the unavailability of appropriate vectors. In the present study, we introduced a novel method inspired by the restriction enzyme-based cloning method with some modifications named All-In-One (AIO) cloning.Materials and Methods: The PCR product and vector for cloning were digested in one 0.2 ml tube with a total volume of 20 µl. Completely digested products were checked and inactivated by heat treatment. Digested genes and vectors were directly used for the ligation step in this 0.2 ml tube, without any purification step required. Finally, ligation products were transformed into competent E. coli DH5α by the heat shock method.Results: More than eight different clones were generated by using AIO cloning, which all the necessary reactions were performed in one single 0.2 ml tube. This method was efficient in cloning a wide range of DNA fragments, from 200 to 1300 bps.Conclusions: Collectively, AIO provided an alternative yet sufficient cloning protocol, reducing the loss of DNA components, and saving materials, labor and time, especially where research materials were not abundantly available. 
  • Immunoinformatics Approach for Glycoprotein B-based Vaccine Candidate
           Design Against Infectious Laryngotracheitis Virus

    • Abstract: Introduction: Infectious Laryngotracheitis Virus (ILTV) poses a highly contagious upper respiratory tract illness and is regarded as a major concern for the poultry industry worldwide. Considering the emergence of novel virulent variants of ILTVs, the development of novel vaccines is needed to control the disease. This study was carried out based on immunoinformatics and vaccinology strategies to introduce an effective ILTV vaccine candidate targeting glycoprotein B (gB).Materials and Methods: Both T-cell and B-cell epitopes of the gB protein with the potential to induce immune responses were identified. The highly antigenic and immunogenic PH-1 domain was selected as the potential vaccine candidate. The physiochemical properties and tertiary structure model of the domain were predicted, refined, and validated. The resulting high‐quality model was applied for docking analyses with toll‐like receptor 2. The affinity of the vaccine candidate to bind with the immune receptor and generate the appropriate immune responses was calculated based on the free energy level.Results: The results indicated the PH-1 domain of ILTV gB protein to be immunogenic and was effective in stimulating the T helper, T cytotoxic, and B cells. The domain showed high-affinity binding and stability with the TLR2 immune receptor.Conclusions: Collectively, this research provides an immunogenic candidate for designing a protein-based vaccine against ILTV. The efficacy of the construct should be examined in lab settings and in animal models. 
  • Production and Characterization of Tannase By Bacillus subtilis in Solid
           State Fermentation of Corn Leaves

    • Abstract: Introduction: Tannase (tannin acyl hydrolase EC3.1.1.20) is an industrially important enzyme with extensive applications. The current study aimed to optimize the tannase production employing corn leaves as substrate and characterize tannase activity.Materials and Methods: Tannase producing bacterial strains were isolated from Catla catla fish gut. The highest enzyme-producing bacterial strain was identified as Bacillus subtilis using 16S rDNA sequencing.Results: Fermentation parameters and additional medium components were optimized with the application of one variable at a time and enhanced tannase was obtained with 50% substrate moisture, acetate buffer (pH 4.0) as enzyme extraction medium with 2 ml volume, 45 °C incubation temperature, pH 5, 2% inoculum size, 24 h incubation time, 150 rpm agitation, large-sized substrate particles (4.0 mm), enzyme extraction without centrifugation and medium components (MgSO4, 4% tannic acid and yeast extract). The central composite design was employed to optimize the concentrations of optimal medium components, which were found as 4.0% tannic acid, 0.5% MgSO4 and 1.5% yeast extract for the highest tannase production (211.97±0.08 U/ml). Tannase characterization revealed the maximum tannase activity at pH 8, 30 °C (with 30 min incubation period and 0.35% substrate concentration).Conclusions: The results of the present study revealed the potential of utilization of agricultural resources (corn leaves) as a low-cost substrate to reduce the production cost of tannase. 
  • Activity of a Novel Antimicrobial Peptide with Nitric Oxide Induction
           against some Pathogenic Bacteria

    • Abstract: Introduction: Bacterial resistance against antibiotics has caused many problems in treating humans and animal infections worldwide. Nowadays, researchers are continuously seeking to develop novel antibacterial to tackle the issue of microbial resistance. Antimicrobial peptides have been introduced as new effective strategies that kill bacteria quickly and cause less antibiotic resistance. In this study, we evaluated the antibacterial and cytotoxic effects of a synthesized peptide (NRWCFAGRR-NH2) on some Gram-positive and –negative bacteria and eukaryotic cells.Materials and Methods: Twelve bacterial strains were selected to study the antimicrobial effect of the NRWC peptide. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays were used to study the bacteriostatic and bactericidal activity of these peptides, respectively. The cytotoxic effect of the peptide was evaluated on Hela cell line and human RBC using the MTT assay and hemoglobin release measurement, respectively. The J774 macrophage cell line was used to measure nitric oxide production in response to the peptide.Results: The results showed that NRWCFAGRR peptide has a bactericidal and inhibitory effect on all 12 bacterial strains' growth in a dosedependent manner. It has also been proven that the toxic effect of the peptide on human cells is evitable at the MIC and MBC concentration. The highest amount of nitric oxide production was induced after 48 hours of treatment.Conclusions: Considering the research conducted in the field of antimicrobial peptides, our designed peptide has antimicrobial properties that kill some of the pathogenic microorganisms directly and can theoretically kill some organisms indirectly via induction of nitric oxide by macrophages. 
  • Evaluation of the Anti-cancer Effect of Curcumin on MCF-7 Cells in 3D
           Culture Conditions to Increase the Efficacy of Breast Cancer Treatment

    • Abstract: Introduction: Breast cancer is the most common cancer among women. Information published by The Iranian Cancer Research Center in 2019 shows that one of every 10 to 15 women is afflicted with this cancer. As one of the active ingredients of turmeric, curcumin has a wide range of biological properties, such as antioxidant and anti-cancer activity. This study aimed to evaluate the anti-cancer effects of curcumin on breast cancer cells in 3-Dimensional (3D) culture conditions.Materials and Methods: To achieve a 3D environment, we used encapsulation of cells in alginate hydrogel. The anti-cancer effects of curcumin at concentrations of 20, 40, and 80 μM on MCF-7 breast cancer cells in 3D culture were evaluated by MTT, neutral red, comet assay, cytochrome c, Nitric Oxide (NO), catalase, and glutathione assays. The culture medium was used as the negative control and the cell-containing medium was used as the positive control. Data were analyzed by two-way ANOVA using GraphPad InStat software, and the significance was considered at the level of P<0.05.Results: Curcumin reduces the production of cellular NO and increases the production of catalase and glutathione, which confirms the results of the NO test. In addition, the release of cytochrome c from Mitochondria from cells treated with different concentrations of curcumin compared to control cells are significant. The evaluation of the toxicity effect of curcumin at concentrations of 20, 40, and 80 μM using comet assay showed that this substance induces apoptosis in MCF-7 cells in a dose-dependent manner.Conclusions: The findings of this study showed that the anti-cancer effect of curcumin on MCF-7 cells under 3D culture conditions could increase the effectiveness of treatment. The Cell survival rate actually depended on curcumin concentration. 
  • Evaluation of IgM-based ELISA for the Detection of Strongyloidiasis by NIE

    • Abstract: Introduction: Strongyloidiasis is a neglected disease caused by Strongyloides stercoralis. Fast and robust detection of this parasite can avoid its auto-infection cycle and therefore from the high parasitic load. The culture of stool and stool-based microscopy techniques are the most used methods for parasite diagnosis; however, these methods are not sensitive enough. Immunological methods are more sensitive diagnostic tools and can be employed for the detection of this parasite. Since IgG4 immunoreactive protein (NIE) is a major pathogenicity factor of the parasite, in the present study, an IgM-ELISA method was developed to investigate the efficiency of anti-NIE IgM antibodies in the detection of Strongyloides stercoralis.Materials and Methods: For this aim, 50 serum samples were gathered from positive patients along with 50 serum patients from healthy people. IgM antibodies were detected by using the ELISA technique and the data were analyzed by statistical analyses.Results: Statistical analyses showed that IgM-ELISA was able to diagnose the disease with the sensitivity and specificity of 65.0%.Conclusions: The developed IgM-ELISA method was relatively but not sufficiently successful at a robust diagnosis of strongyloidiasis. However, the sensitivity and specificity of this method were not good enough to be considered a reliable test for the diagnosis of the disease compared to the IgG- ELISA. 
  • Increase the Efficiency of MKN45 Cell Line to CD44 Editing by CRISPR/Cas9:
           A Hypothesis About P53 Suppression in Gene Editing

    • Abstract: The clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) used for genome editing. The usage of CRISPR-Cas9 in gene editing is faced with certain limitations including off-target mutation, decreased homologous recombination (HR) repair, and immune system responses. It seems that if Cas9 expressed in an inducible manner, off-target mutations may decrease. P53 decreases the activity of the HR pathway in the cell cycle, so, the decrease in P53 level may increase the activity of this pathway. Based on this topic, for the first time, we designed ''px601-Turbo GFP-TRE-shRNA P53'' as a CRISPR-based vector. The use of this vector can simultaneously induce expression of Cas9 and shutdown transiently P53 under an inducible promoter and an inducing agent. Therefore, shutdown transiently P53 may be leading to reduced off-targets and increased accuracy of genome editing. In the human gastric cancer MKN45 cell line, the P53 gene expresses at a normal level. Also, CD44 in this cell line has overexpression and is a gastric cancer stem cell marker. To evaluate this hypothesis, CD44 will be targeted for a specific sequence change (editing) by the px601-Turbo GFP-TRE-shRNA P53 vector. Accordingly, after cloning and virus preparation, MKN45 cell lines will be transduced in the presence of the appropriate doxycycline (DOX) dosage. Ultimately, to evaluate the vector efficiency, DNA extraction and whole-genome sequencing (WGS) will be done and compared with the transduced MKN45 cells without an inducible prompter and DOX as control. Also, the Sanger sequencing for the target gene must be done. This temporary inducible expression may appear to increase the efficiency of the CD44 gene editing and reduce off-targets.
  • An Epidemiological Review on Toxoplasma Prevalence in Sheep and Goat Meat
           in Iran

    • Abstract: There are limited parasites that can infect both humans and animals, and Toxoplasma gondii is among those limited ones. Its primary host is cat, but it can infect warm-blooded animals like humans, goats, sheep, cattle, dogs, etc. Although the main transmission route in humans is via food, including raw or undercooked meat, the infection route in animals is via mother to child and ingesting sporulated oocysts. Due to the dangerous results of this protozoan, including abortion, stillbirth, different degrees of mental or physical retardation, prevention of such infection has to be seriously considered. Cattle have a natural resistance against Toxoplasma infection. Therefore, its prevalence has more importance in goats and sheep. According to the studies that have measured Toxoplasma gondii infection and prevalence on world- and country-wide scale, infection of this protozoan is highly related to the geographic status, susceptible animals, potential hosts, and eating habits. In the present paper, we review the prevalence, epidemiology spectrum of T. gondii in animals in Iran. This knowledge should be useful to biologists, public health workers, physicians, and veterinarians.
  • Production and Purification of CAMP-Sialidase Chimer Protein as a Vaccine
           Candidate for Acne and Its Immunization in the Animal Model

    • Abstract: Introduction: Acne vulgaris affects ~85% of young adults aged 12–25 years. Regarding the critical role of Propionibacterium acnes in the pathogenesis of acne and current therapeutic failures, developing efficient acne vaccines are appreciated. The purpose of this study was to design a chimeric protein from CAMP and sialidase parts of Propionibacterium acnes and evaluating its immunogenicity in a mice model as an acne vaccine candidate. Materials and Methods: CAMP-Sialidase recombinant gene expression was carried out through cloning in the vector pET28a and transferring to E.coli BL21DE3. The protein was purified using the Ni-NTA column and its concentration was measured. The recombinant protein was injected in test and control mice groups. Then, antibody titration and challenge tests were made to determine the immunogenicity. Results: After successful expression and purification, the protein band was observed at a molecular weight of 65 kDa. Western blotting confirmed the purified protein. The results of serum ELISA indicated the IgG titer was 1:204800 and 1:1600 against the recombinant protein and inactivated P. acne, respectively. Although there was no change in test mice, inflammation happened in 50% of the control group. Conclusions: The current study demonstrated that the CAMP-Sialidase recombinant protein can appropriately induce humoral antibodies. However, more evaluations need to introduce it as an acne vaccine candidate.
School of Mathematical and Computer Sciences
Heriot-Watt University
Edinburgh, EH14 4AS, UK
Tel: +00 44 (0)131 4513762

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